Stabilizing clinical samples for prolonged ambient storage is of outmost importance to reduce operational costs in the rapidly evolving field of translational research. High-throughput clinical proteomics issues stringent requirements fo analyte quality that are not currently met by ambient preservation methods. It is therefore necessary to develop new, cost-efficient storage methods for plasma protein preservation tailored towards clinical proteomic applications. This application proposes t investigate room-temperature storage of dried, nitrogen flushed plasma spots (DPS-n) on filter paper substrates as a viable alternative to snap-freezing and cryostorage for the stabilization and preservation of many plasma proteins. The low sampling volume required for DPS-n is particularly attractive in pediatric applications, where blood draws are sized to the age of the patient. Therefore, all work will be performed on plasma obtained from consenting pediatric patients. The first part of the described approach (Specific Aims 1) focuses on gaining insights into the protein aging process involved in DPS-n to evaluate mid- to long-term room temperature storage of human plasma proteins on filter substrates over a period of 2 years. For all identified proteins, we will monitor over time the amino-acid modificatins that are due to active, ex-vivo mechanisms of protein degradation, such as proteases and reactive oxygen species. Several chemical and non-chemical avenues of protein stabilization will be explored to propose optimized protocols for protein preservation usin this method. The second part of our approach (Specific Aims 2) evaluates the potential of DPS-n to discern differentially expressed proteins by comparing protein profiles of pediatric patients with known diagnosis versus healthy volunteers in plasma preserved using the optimized conditions determined in the first aim of this application. We will compare ur results against snap-frozen, cryopreserved plasma to determine potential trends in differentially expressed proteins related to preferential preservation of specific proteins fr one method. Key strength of this proposal are the comprehensive characterization of conditions influencing protein stability and the validation of DPS-n in a clinical stud on differentially expressed protein to lay the foundation for a widespread use of this method in translational studies. We expect that our findings will significantly advance the fields of biopreservation and biobanking by validating and optimizing dried plasma spotting as a viable long term storage strategy for high- throughput proteome analysis and biomarker development studies. Both aims will leverage the state-of-the art analytical capabilities of a world-class translational omics institute under the supervision of a collaborative team of domain experts in mass spectrometry and pediatric healthcare.